Amperometric method for the quantitative determination of 1,4-dihydronicotinamide adenine dinucleotide (nadh) in solution

ABSTRACT

A method is disclosed for the quantitative determination of 1,4-dihydronicotinamide adenisne dinucleotide (NADH) in solution. The method comprises contacting the NADH-containing solution with an activated carbon electrode, maintaining the carbon electrode at a controlled, fixed potential effective to cause oxidation of NADH at the electrode surface, and measuring the current output from the carbon electrode, wherein there is used a noble metal containing preferably a platinized or palladized activated carbon electrode comprising a porous, heterogeneous, resin-bonded layer of activated carbon or graphite particles comprising the finely divided noble metal preadsorbed thereon and bonded together with a natural or synthetic resin binder, preferably a hydrophobic resin such as polytetrafluoroethylene.

This invention relates to a method for the quantitative determination of1,4-dihydronicotinamide adenine dinucleotide (NADH) in solution.

NADH and its oxidised counterpart NAD are cofactors in numerous enzymecatalysed redox reactions. In some, an enzyme substrate is oxidised inthe presence of cofactor NAD and a suitable oxidase or dehydrogenase toyield NADH in solution; in others an enzyme substrate is reduced in thepresence of cofactor NADH to yield NAD in solution. In many cases,determination of the NADH concentration can be used as an indicator ofsubstrate concentration, or as a means of following the course of anenzyme reaction involving NADH (or NAD).

It is known that NADH concentration in solution can be measuredcolorimetrically, but colorimetric methods on the whole aredisadvantageous. Much more advantageous are electrochemical methods, butattempts to determine NADH electrochemically have so far not met withany very great degree of success. It is known, for example, that NADHconcentration can be determined by an amperometric assay in which NADHis oxidised at an electrode at a fixed, controlled, potential, thecurrent passing under suitable conditions being proportional to NADHconcentration. Unfortunately the electrochemical oxidation of NADHrequires a high overpotential, and the NADH is generally not oxidisedcleanly at the electrode surface; for example, in many cases the surfaceof the electrode is quickly fouled by formation of a surface film whichaffects the size and speed of the electrochemical response: I. Moirouxand P. J. Elving, J. Amer. Chem. Soc. (1980) 102, 6533-6538, and D. G.Johnson, M. D. Ryan and G. S. Wilson, Analyt. Chem. (1986) 58, 42R.

There have been many attempts to avoid these problems. For example, ithas been proposed to use modified electrodes coated with a layer ofconducting organic salts: J. J. Kulys, Biosensors (1986) 2, 3-13.Alternatively it has been proposed to use an adsorbed redox mediatorsuch as Meldola's blue to couple the oxidation reaction more effectivelyto the electrode and/or to lower the oxidation potential: L. Gorton etal., J. Electroanalyt. Chem. (1984), 161, 103-20. In another proposalredox mediators have been used in free solution. For example, methoxyphenazine methosulphate has been used with a modified pyrolitic graphiteelectrode: Y. Kimura and K. Nihi, Analytical Sciences (1985), 1, 271-4.Other experiments have been carried out with platinum, graphite andglassy carbon electrodes, but as yet no electrochemical method for thedetermination of NADH has been developed which is both rapid andreproducible.

In accordance with the present invention it has been discovered thatNADH can be oxidised cleanly, with good amperometric response, both inbuffer solutions containing NADH alone, and in solutions containingenzyme, enzyme substrate and NADH, using an activated carbon electrodeof a type used in fuel cell technology and comprising a heterogeneousresin-bonded layer of noble metal containing, preferably platinised orpalladised (which terms as used herein include materials containing ortreated with platinum and/or palladium oxide, as well as materialscontaining or treated with platinum or palladium metal) carbon orgraphite particles bonded with a natural or synthetic resin binder,preferably a synthetic, hydrophobic binder, such as a fluorocarbonresin, most preferably polytetrafluoroethylene. Preferably a platinisedor palladised activated carbon electrode is used in which the carbon orgraphite particles are platinised or palladised by adsorbing ordepositing colloidal platinum or palladium metal, or platinum orpalladium oxide, onto the surface of the powder particles beforebonding, the resultant electrode comprising a heterogeneous porousactivated carbon powder layer with colloidal platinum or palladium, orthe corresponding oxides, distributed substantially uniformly throughoutthe layer. The resin-bonded layer of platinised or palladised activatedcarbon or graphite particles may be self-supporting, but will usually besupported by a support member, preferably an electrically conductivesupport member, and preferably a layer of electrically conductive carbonpaper to which the platinised or palladised carbon or graphite particlesare bonded as a surface layer, or impregnated into a carbon fibre web.Whilst the platinised and palladised materials are preferred, othernoble metal containing activated carbon electrodes, e.g. gold containingelectrodes, may be used.

Herein, the terms "platinised" and "palladised" include the oxidesunless the context requires otherwise.

Also herein, the terms "activated" carbon, "activated" graphite, etc.refer to highly porous, high surface area carbon and graphite materialshaving surface areas of 50 m² /g or greater, and more usually in excessof 200 m² /g, e.g. from 200 to 600 m² /g or higher. Such high surfacearea materials are obtained, for example, by heat treatment of carbon orgraphite powders in steam or CO₂ to give a high surface area productgenerally referred to in the art as "activated carbon".

Quite apart from the stability, reproducibility, and rapid responsetimes already mentioned, a further particular advantage of the presentmaterials is that they can be used to monitor NADH concentrations atrelatively low potentials, e.g. in the range 0 to 600 mV or even atnegative potentials with reference to the standard Ag/AgCl referenceelectrode, as against the 750 mV and upwards required to monitor NADHconcentrations using glassy carbon or graphite electrodes. The presentelectrodes are thus characterised by relatively low background current,and hence improved sensitivity. The electrodes are also characterised bytheir low response to potentially interfering species, such as uricacid, frequently present in biological or clinical samples.

The preferred electrode substrates used in accordance with thisinvention are, in fact, commercially available materials sold by thePrototech Company of Newton Highlands, Mass., and used heretofore aselectrocatalytic gas diffusion electrodes in fuel cells. The preparationof such materials is described in detail in U.S. Pat. No. 4,044,193,U.S. Pat. No. 4,166,143, U.S. Pat. No. 4,293,396 and U.S. Pat. No.4,478,696, to which reference should be made for full details. In broaddetail, however, colloidal platinum with a particle size in the range 15to 25 Angstroms (1.5 to 2.5 nm) is adsorbed onto the surface of powderedcarbon (particle size 50 to 300 Angstroms:5 to 30 nm), for example, byformation of a platinum sol in situ in the presence of powdered carbonwhich acts as a nucleating agent for the sol. The platinised carbonparticles are then moulded onto an electrically conductive supportingstructure, e.g. electrically conductive carbon paper, using a syntheticresin binder, preferably a fluorinated hydrocarbon resin, and especiallypolytetrafluoroethylene. Alternatively, platinum or palladium oxidehaving a similar particle size range may be used in place of thecolloidal platinum, and adsorbed onto the carbon or graphite particlesin a similar manner.

In an alternative, disclosed in U.S. Pat. No. 4,293,396, the platinisedcarbon particles are impregnated into a preformed porous carbon clothand bonded therein using the fluorocarbon resin, preferablypolytetrafluoroethylene. It is to be understood, however that thepresent invention is not limited to the use of Prototech materials, butembraces other similar substrate materials comprising a porousresin-bonded layer of platinised or palladised, or other noble metalcontaining activated carbon or graphite particles.

Whilst the preferred resin binders used to bind the platinised orpalladised carbon or graphite particles are hydrophobic fluorocarbonresins, particularly polytetrafluoroethylene, other suitable natural orsynthetic resin binders may be used, for example polyethylmethacrylate,polyvinyl acetate, polyvinyl chloride, polycarbonates,poly(4-methylpentene-1) polyisoprene, isoprene, polychloroprene,poly(1,3-butadiene), silicone rubber and gelatin.

The proportion of binder to the noble metal containing activated carbonor graphite particles, on a weight basis, may range from 10 to 75%binder and 90 to 25% activated carbon or graphite, preferably 20 to 50%binder and, correspondingly, 80 to 50% activated carbon or graphite. Theloading of noble metal, e.g. platinum or palladium or theircorresponding oxides, or gold, on the activated carbon or graphiteparticles may range from 1 to 10% based on the total weight of activatedcarbon or graphite and binder, preferably from 2 to 8%, most preferablyfrom 4 to 6%.

Instead of moulding the resin/activated platinised or palladised carbonpowder directly onto the surface of a suitable support, e.g. directlyonto the surface of electrically conductive carbon paper, the mixture ofbinder and platinised or palladised carbon powder may be suspended in asuitable inert medium, and applied to the surface of the substrate by ascreen printing technique, thereby providing a thin film of resin-bondedplatinised or palladised carbon particles on the surface of thesubstrate.

As well as the direct quantitative measurement of NADH in solution, theelectrodes and process of the present invention may be used in thequantitative determination of NADH generated or consumed in situ forexample by the enzymatic reaction between an enzyme and its cofactor.Such reactions include for example the conversion of pyruvate to lactateby lactate dehydrogenase, i.e. the reaction ##STR1## which reaction maybe monitored by the decrease in NADH concentration; and the oxidation ofglucose to gluconolactone by the reaction ##STR2## which can bemonitored by the increase in NADH concentration as the reactionproceeds. To this end the activated platinised or palladised carbonelectrodes used in accordance with this invention may have an enzymesuch as lactate dehydrogenase or glucose dehydrogenase incorporated intoor immobilised onto the resin-bonded carbon layer by any of the enzymeimmobilization techniques known in the art and taught for example inEP-A-0 247 850.

In a further modification of this concept the present invention alsoenvisages a one off, disposable enzyme electrode and method in which theenzyme electrode itself comprises not only the immobilised enzyme, butalso the appropriate cofactor for that enzyme, either NAD or NADH as thecase may be, the enzyme electrode thus having the capability ofresponding amperometrically to the activity of the enzyme, as determinedby the change in NADH concentration, when in contact with a sample, e.g.a clinical or biological sample, containing the relevant substrate forthat enzyme, irrespective of whether that sample contains the necessarycofactor, since that is supplied by the electrode itself. The NAD orNADH cofactor may be incorporated into the electrode in any suitablemanner such as impregnation with a suitable solution of either NAD orNADH and drying.

As is also well known in the art, the surface of the electrode materialmay or may not be protected by a porous membrane, such as apolycarbonate film having a pore size of for example about 0.03 μm.Other suitable membrane materials may also be used.

The invention is further described with reference to the accompanyingdrawings, in which:

FIG. 1 is a diagrammatic section through a modified Rank Brotherselectrochemical cell used to test the NADH response of the activatedcarbon electrodes in accordance with this invention;

FIG. 2 shows the electrode response to successive additions of NADH tothe cell using a platinised carbon paper (PCP) electrode according tothis invention;

FIG. 3 shows the response of the PCP electrode to NADH at variouspoising potentials versus the Ag/AgCl reference electrode;

FIG. 4 shows the response of the electrode to pyruvic acid in thepresence of lactate dehydrogenase (LDH);

FIG. 5 shows the response of the electrode to acetaldehyde in thepresence of alcohol dehydrogenase (ADH);

FIG. 6 is another graph showing the response of platinised carbon paperelectrodes to NADH concentration in accordance with this invention;

FIG. 7 shows the results of another experiment involving the response ofthe electrode to pyruvic acid;

FIG. 8 shows the response of the electrode to NADH produced in situ bythe enzymatic oxidation of glucose using glucose dehydrogenase;

FIG. 9 shows the similar response curve for a platinum oxide containingelectrode; and

FIG. 10 shows the response curve for a palladised activated carbonelectrode.

In the following Examples, various platinised or palladised carbon paper(PCP) electrodes were tested for their response to NADH in a modifiedRank oxygen electrode system (Rank Brothers, Bottisham, Cambridge) andas shown in the accompanying drawings (FIG. 1). The modified Rank cellsystem comprises a two-part cell having a base (1) and an annular jacket(2) enclosing a water chamber (h), through which water may be circulatedto control the temperature of the cell, the two parts being connectedtogether by the captive threaded collar (3). Centrally located in thebase (1) is a platinum contact button (d) onto which is placed the testdisc (a) of paper electrode material and which is held in place on theplatinum contact by rubber O-ring seals (e) and (f) when the two partsof the cell are coupled together.

Inserted into the top of the cell, which of course will contain the testNADH-containing solution, is a stopper (4) supported by an adjustablecollar (g) and in which are mounted a platinum counter electrode (b) andan Ag/AgCl reference electrode (c). The tests were carried out with theworking electrode poised at various potentials in the range 100 to 600mV with reference to the Ag/AgCl electrode. Other tests were carried outin a two electrode cell as illustrated in FIG. 16 of EP-A-0 247 850 andas described therein in detail. In the two electrode cell embodiment,the electrode material is held against a platinum contact button in thebase of the cell by means of a polycarbonate (0.03 μm pore size)membrane and to which the NADH containing sample is applied. Surroundingthe platinum contact, in the base of the cell, but separated therefromby an insulating sleeve is an annular Ag/AgCl reference electrode. Theelectrode cell is polarised at various potentials relative to theAg/Ag/Cl electrode and the output current monitored at variouspotentials.

The invention is illustrated by the following Examples in which theelectrode material is a platinised carbon paper (PCP) as supplied by thePrototech Company of Newton Highlands, Mass. and developed by them asgas diffusion electrodes. The PCP electrode material is preparedaccording to the teachings of U.S. Pat. No. 4,044,193 by initiallyplatinising carbon powder particles (Vulcan XC-72), nominal particlesize 30 nm) by the oxidative decomposition of a complex platinum sulfiteacid in the presence of the carbon powder using H₂ O₂, thereby todeposit colloidal platinum, particle size 1.5 to 2.5 nm, on the surfaceof the carbon powder particles. Following platinisation, the platinisedcarbon powder is subsequently moulded and bonded onto the surface of acommercial, graphitised electrically conducting carbon paper usingapproximately 50% by weight, based on platinised carbon powder, ofpolytetrafluoroethylene as the binder. The resulting platinised carbonpaper electrode material has a thickness in the range 0.1 to 0.5 mm, anda platinum loading of 0.24 mg.cm⁻². For the purpose of the followingtests (Examples 1 to 3), the carbon paper electrode material was cutinto 5 mm diameter discs and mounted on the platinised working electrodeof the cell system shown in FIG. 1 of the accompanying drawings. Theactual area of the carbon paper electrode exposed to the sample in eachcase is approximately 0.16 cm².

The results obtained are as follows:

EXAMPLE 1 Electrochemical Oxidation of NADH on Platinised Carbon Paper(PCP)

Using a standard potentiostatic technique, samples of a 20 mM NADHsolution in Tris/HCl pH 9 buffer were added to the cell containing 2 mlof 0.1M pH 7 phosphate/1M KCl buffer solution. The platinised carbonpaper electrode (PCP) was poised at various potentials with respect tothe Ag/Ag/Cl reference electrode. The counter electrode was platinum.Stable current plateaus were obtained (FIG. 2) which were proportionalto NADH concentration (Table 1 and FIG. 3).

                  TABLE 1                                                         ______________________________________                                        Current response to platinised carbon paper                                   to NADH at various poising potentials                                                       Current Output in μA at                                      NADH Concentration/mM                                                                         100 mV    300 mV   600 mV                                     ______________________________________                                        0.95             7        15       28                                         1.8             13        25       53                                         2.7             --        32       73                                         4.0             38        42       113                                        6.6             --        52       183                                        10.0            60        --       --                                         ______________________________________                                    

EXAMPLE 2 Response of NADH-Electrode System to Pyruvic Acid in thePresence of Lactate Dehydrogenase (LDH)

A similar cell was set up containing 12.5 mM NADH and 10 mM pyruvic acidin 2 ml of pH 7 phosphate/KCl buffer at 25° C. The working, counter andreference electrodes were as in Example 1. The working electrode waspoised at 400 mV and gave a steady signal of 170 μA above background. Onaddition of 120 units of LDH (bovine heart Type XV) the currentdecreased at an initial rate of 92 μA/min (FIG. 4) showing that theenzymatic conversion of pyruvate to lactate is efficiently monitored viathe agency of NADH electrochemically coupled to the electrode.

EXAMPLE 3 Response of NADH-electrode System to Acetaldehyde in thePresence of Alcohol Dehydrogenase (ADH) on a PCP Electrode

The cell was set up containing 3 mM NADH and 35 mM acetaldehyde in 2 mlof pH 9 Tris/HCl buffer at 25° C. The working, counter and referenceelectrodes were as in Example 1. The working electrode was poised at 400mV and gave a steady signal of 40 μA above background. On addition of 2units of ADH (equine liver) the current decreased at an initial rate of130 μA/min (FIG. 5) showing that the enzymatic conversion ofacetaldehyde to ethanol is efficiently monitored via the agency of NADHelectrochemically coupled to the electrode.

In the following examples either a two electrode cell or a threeelectrode cell configuration was employed. The three electrode cell wasas herein described and illustrated in FIG. 1. The two electrode cellwas identical in construction to that shown in FIG. 16 of EP-A-0 247 850to which reference should be made for further details.

EXAMPLE 4 (FIG. 6)

The data was compiled using the two electrode configuration polarised at200 mV. A buffer of 16 mmol/L NaH₂ PO₄, 53 mmol/L Na₂ HPO₄, 52 mmol/LNaCl, 1.5 mmol/L ethylene diamine tetraacetic acid, pH 7.4, was used.After achieving a stable background current in this buffer, the bufferwas wiped off the membrane and replaced with samples of NADH in the samebuffer. The peak current was recorded. FIG. 6 shows the responses from aplatinised carbon paper electrode as commercially available from thePrototech Company and comprising resin-bonded platinised carbonparticles deposited on an electrically conductive carbon paper backingsheet, the resin-bonded platinised carbon layer comprising, on a weightbasis, 50% polytetrafluoroethylene, 45% finely divided carbon (VulcanXC72) and 5% colloidal platinum preadsorbed onto the carbon powder. Tominimise the background current a 5 mg/ml protein solution (glucoseoxidase) was adsorbed onto the electrode overnight prior to NADHmeasurements. It should be noted that the glucose oxidase is merely anexample of a suitable protein.

EXAMPLE 5 (FIG. 7)

This illustrates the applicability of the invention to measurement ofthe substrates of NADH-utilizing enzymes. A three electrode cell wasused as previously described but equipped with a magnetic stirrer bar.The working electrode was platinised carbon paper as in Example 4, butL-lactate dehydrogenase (EC 1.1.1.27 from beef heart) was immobilised tothe electrode via carbodiimide coupling, see EP-A-0 247 850, but using a1 mg/ml solution of lactate dehydrogenase (from Sigma Chemicals, typeXV, 500 units per mg protein). The cell contained 12.5 mmol/L NADHinitially, in 0.1 mol/L phosphate/l mol/L KCl buffer, pH 7. Thepolarising potential was 350 mV. The apparatus could be used to monitorthe consumption of NADH when aliquots pyruvic acid were added to thecell, as shown in the FIGURE.

EXAMPLE 6 (FIG. 8)

Example 5 was repeated except that the immobilised lactate dehydrogenasewas replaced by glucose dehydrogenase (EC 1.1.1.47 from Bacillus spp,supplied by Sigma, 100-300 U/mg protein) immobilised onto the electrodein a similar manner. Whereas lactate dehydrogenase is used to measurepyruvic by monitoring NADH consumption

    pyruvate+NADH→lactate+NAD

the glucose dehydrogenase is used to measure glucose by following NADHproduction:

    β-D-glucose+NAD→D-gluconolactone+NADH

The cell contained 0.1 mol/L phosphate/0.1 mol/L KCl/2.4 mmol/L NAD, pH7.

EXAMPLE 7 (FIG. 9)

Following the procedure outlined in Example 4, the current output of aplatinum oxide containing carbon electrode is measured at 200 mV(against a Ag/AgCl reference electrode) in a two electrode cell atvarious NADH concentrations, and shows a substantially linear responseequivalent to that of the platinised carbon paper electrodes. In thiscase the electrode material comprises a layer of resin-bonded(polytetrafluorethylene) carbon particles (Vulcan XC72) having 5% byweight (based on total weight of the resin-bonded particles) platinumoxide preadsorbed onto the carbon powder particles; binder 50% byweight, carbon 45% by weight, and bonded to the surface of electricallyconductive Toray (trade mark) carbon paper.

EXAMPLE 8 (FIG. 10)

Once again, following the procedure outlined in Example 4, the currentoutput of a palladised carbon paper electrode is measured at 200 mV(against Ag/AgCl) using the two electrode cell configuration at variousNADH concentrations. Once again the response (FIG. 10) is substantiallylinear. The electrode material is as described in Example 7 save thatthe resin-bonded carbon particles comprise 5% by weight preadsorbedfinely divided palladium.

The above Examples demonstrate the use of the electrode materials toproduce a rapid and reproducible oxidation of NADH. These responses arein marked contrast to those given by most other electrode materials, asdemonstrated in comparable experiments using platinum, glassy carbon, orgraphite electrode materials, which (with rare exceptions) are generallysluggish, relatively insensitive, and of much poorer reproducibility.The effectiveness of the platinised or palladised carbon electrodes wehave used appears to be a result of their particular heterogeneousstructure and its compatibility with biological molecules such as NADHand enzymes. The oxidation of NADH also proceeds efficiently in thepresence of enzymes and substrates, and can be used as a basis for rapidNADH-coupled enzymatic assays. The potential for efficient assay ofpyruvate (using LDH) and acetaldehyde (using ADH) is clear from theabove Examples, but a very large number of similar assays are alsoaccessible using other enzymes and substrates.

Although the invention has been described herein solely with referenceto the determination of NADH in solution, phosphorylated1,4-dihydronicotinamide adenine dinucleotide (NADPH), i.e.phosphorylated NADH, may be determined by exactly the same technique.Moreover, since NADPH (or NADP) is a cofactor in a selected group ofenzyme catalysed reactions, rather than NADH (or NAD), the technique ofthis invention enables such reactions to be monitored in exactly thesame way, i.e. by amperometrically determining either the consumption orthe production of NADPH in solution. Thus all references herein to NADH,or NAD, are to be taken to include the phosphorylated derivative unlessthe context requires otherwise.

We claim:
 1. A method for the quantitative amperometric determination ofNADH in solution in a liquid amenable to measurable electrochemicalaction of NADH therein, which comprises:(a) contacting a sample of saidliquid with an electrode, said electrode comprising in contact with thesample a porous layer of finely divided activated carbon or graphiteparticles having finely divided platinum or palladium adsorbedthereonto, said particles being bonded together in said layer by aresin; (b) maintaining said electrode at a potential effective to causeoxidation of NADH in the sample; and (c) measuring the current produced.2. A method for the quantitative amperometric determination of acompound selected from the group consisting of NADH, oxidized NADH(NAD), phosphorylated NADH (NADPH) and oxidized NADPH (NADP) in solutionin a liquid amenable to measurable electrochemical action of saidcompound therein, which comprises:(a) contacting a sample of said liquidwith an electrode, said electrode comprising in contact with the samplea porous layer of finely divided activated carbon or graphite particleshaving finely divided noble metal or noble metal oxide adsorbedthereonto, said particles being bonded together in said layer by aresin; (b) maintaining said electrode at a potential effective to causechange of the state of oxidation of said compound in the sample; and (c)measuring the current produced.
 3. A method according to claim 2,wherein liquid being a clinical liquid containing NADH or NADPH.
 4. Amethod according to claim 1 or 2, said electrode comprising anelectrically conductive support member having said layer formed thereonas a surface layer of said resin-bonded particles.
 5. A method accordingto claim 4, said support member being an electrically conductive carbonpaper.
 6. A method according to claim 2, said compound being NAD orNADH.
 7. A method according to claim 1 or 2, said resin being afluorocarbon resin.
 8. A method according to claim 1 or 2, said resinbeing polytetrafluoroethylene.
 9. A method according to claim 1 or 2,said activated carbon or graphite particles having particle sizes in therange of about 5 to 30 nm, and having colloidal particles of platinum orpalladium adsorbed thereonto prior to being bonded together, and beingbonded together by a fluorocarbon resin.
 10. A method for amperometricdetermination of the quantity in solution in a liquid of a substancethat will undergo enzymatic reaction in the liquid in the presence of anenzyme capable of catalyzing said reaction when NADH is present in thesolution as a cofactor to be oxidized electrochemically concomitantlywith the enzymatic reaction, which method comprises:(a) contacting asample of the liquid, in the presence of both said enzyme and saidcofactor, with an electrode maintained at a fixed potential effective tocause oxidation of NADH in the sample, said electrode comprising incontact with the sample a porous layer of activated carbon or graphiteparticles having finely divided platinum or palladium adsorbedthereonto, said particles being bonded together in said layer by aresin, (b) thereby initiating the enzymatic reaction of said substancewith concomitant oxidation of NADH in the sample; (c) measuring thecurrent produced; and (d) determining from the measured current theconcentration, or change of concentration, of NADH in the sample as aresult of the enzymatic reaction, whereby to determine indirectly theamount of said substance present in the sample.
 11. A method foramperometric determination of the quantity in solution in a liquid of asubstance that will undergo enzymatic reaction in the liquid in thepresence of an enzyme capable of catalyzing said reaction when acompound selected from the group consisting of NADH, oxidized NADH(NAD), phosphorylated NADH (NADPH) and oxidized NADPH (NADP) is presentin the solution as a cofactor to be reacted electrochemicallyconcomitantly with the enzymatic reaction, which method comprises:(a)contacting a sample of the liquid, in the presence of both said enzymeand a said cofactor, with an electrode maintained at a fixed potentialeffective to cause electrochemical change of the state of oxidation ofsaid cofactor in the sample, said electrode comprising in contact withthe sample a porous layer of activated carbon or graphite particleshaving finely divided noble metal or noble metal oxide adsorbedthereonto, said particles being bonded together in said layer by aresin, (b) thereby initiating the enzymatic reaction of said substancewith concomitant change of the state of oxidation of said cofactor inthe sample; (c) measuring the current produced; and (d) determining fromthe measured current the concentration, or change of concentration, ofcofactor in the sample as a result of the enzymatic reaction, whereby todetermine indirectly the amount of said substance present in the sample.12. A method according to claim 11, said compound being NAD or NADH. 13.A method according to claim 1, 2, 10 or 11, said electrode being aworking electrode in an electrochemical cell that comprises also acounterelectrode in contact with said sample and a reference electroderelative to which the working electrode is poised at a fixed potential.14. A method according to claim 13, said working electrode beingmaintained at a potential of less than 600 mV relative to said referenceelectrode.
 15. A method according to claim 10 and 11, said enzyme beingimmobilized or adsorbed onto said resin-bonded particles of said layerprior to contact of the sample with said layer.
 16. A method accordingto claim 10 or 11, said cofactor being adsorbed into said layer prior tocontact of the sample with said layer so that cofactor in effectiveamount is supplied from the electrode into the sample when the sample isin contact with the electrode.
 17. A method according to claim 15, saidcofactor being adsorbed into said layer prior to contact of the samplewith said layer so that at least cofactor in effective amount issupplied from the electrode into the sample when the sample is incontact with the electrode.
 18. A method according to claim 10 or 11,said electrode comprising an electrically conductive support memberhaving said layer formed thereon as a thin surface layer of saidresin-bonded particles; said activated carbon or graphite particleshaving sizes in the range of about 5 to 30 nm, having colloidal platinumparticles pre-adsorbed thereon, and being bonded together by afluorocarbon resin.
 19. A method according to claim 18, said supportmember being an electrically conductive carbon paper.
 20. An enzymeelectrode for use in amperometric determination of the quantity in aliquid sample of a substance that will undergo enzymatic reaction in thesample in the presence of an enzyme capable of catalyzing said reactionwhen also in the presence of NADH in solution as a cofactor to beoxidized electrochemically concomitantly with the enzymatic reaction,said electrode comprising(a) a porous layer of finely divided activatedcarbon or graphite particles having finely divided platinum or palladiumadsorbed thereonto, said particles being bonded together in said layerby a resin; (b) a said enzyme adsorbed or immobilized in said layer; and(c) NADH adsorbed in said layer so as to be supplied therefrom intosolution in the sample, for oxidation in the sample concomitantly withthe enzymatic reaction of said substance therein, when the sample is incontact with said layer and an electrical potential is applied throughsaid electrode, whereby electrochemical change of the state of oxidationof said cofactor may be sensed via the electrode to indicate indirectlythe concentration of said substance in the sample.
 21. An enzymeelectrode for use in the aperometric determination of the quantity in aliquid sample of a substance that will undergo enzymatic reaction in thesample in the presence of an enzyme capable of catalyzing said reactionwhen also in the presence in solution of a cofactor, selected from thegroup consisting of NADH, oxidized NADH (NAD), phosphorylated NADH(NADPH) and oxidized NADPH (NADP), to be oxidized or reducedelectrochemically concomitantly with the enzymatic reaction, said enzymeelectrode comprising(a) a porous layer of finely divided activatedcarbon or graphite particles having finely divided noble metal or noblemetal oxide adsorbed thereonto, said particles being bonded together insaid layer by a resin; (b) a said enzyme adsorbed or immobilized in saidlayer; and (c) a said cofactor adsorbed in said layer so as to besupplied therefrom into solution in the sample for electrochemicalreaction in the sample concomitantly with the enzymatic reaction of saidsubstance therein when the sample is in contact with said layer and anelectrical potential is applied through said electrode, wherebyelectrochemical change of the state of oxidation of said cofactor may besensed via the electrode to indicate indirectly the concentration ofsaid substance in the sample.
 22. An enzyme electrode according to claim20 or 21, said electrode comprising an electrically conductive supportmember having said layer formed thereon as a thin surface layer of saidresin-bonded particles; said activated carbon or graphite particleshaving sizes in the range of about 5 to 30 nm, having colloidal platinumparticles adsorbed thereon, and being bonded together by a fluorocarbonresin.
 23. An enzyme electrode according to claim 22, said supportmember being an electrically conductive carbon paper.
 24. An enzymeelectrode according to claim 21, said adsorbed cofactor being NAD orNADH.